EP0508472A2 - Verfahren zur Herstellung von Hybridomen - Google Patents

Verfahren zur Herstellung von Hybridomen Download PDF

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EP0508472A2
EP0508472A2 EP92106282A EP92106282A EP0508472A2 EP 0508472 A2 EP0508472 A2 EP 0508472A2 EP 92106282 A EP92106282 A EP 92106282A EP 92106282 A EP92106282 A EP 92106282A EP 0508472 A2 EP0508472 A2 EP 0508472A2
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Prior art keywords
monoclonal antibody
class
hybridoma
producing
hybridomas
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French (fr)
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EP0508472B1 (de
EP0508472A3 (en
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Katsumi Fujiwara
Kenya Shitara
So Ohta
Nobuo Hanai
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KH Neochem Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • This invention relates to a method of producing hybridomas which comprises converting (class-switching) a hybridoma capable of producing a monoclonal antibody belonging to the IgM class to a hybridoma capable of producing a monoclonal antibody belonging to the IgG class.
  • the monoclonal antibodies of the IgG class as produced by said hybridomas can be used in the treatment of various diseases more widely as compared with monoclonal antibodies of the IgM class.
  • Antibodies are classifiable into the IgA, IgM, IgG, IgD and IgE classes. Furthermore, mouse antibodies of the IgG class can be classified into four subclasses, G1, G 2a , G 2b and G3 (for human antibodies, G1, G2, G3 and G4). When animals are immunized with an antigen, most of the antibodies obtained belong to the IgM or IgG class. IgG molecules have a dimeric structure with a molecular weight of about 160,000 and are relatively easy to handle.
  • IgM molecules are much greater and have a J chain-bridged complicated pentameric structure with a molecular weight of about 900,000, and therefore they have drawbacks: for instance, they are difficult to purify and are difficult to store because of marked tendency toward aggregation, they are readily deactivated due to partial degradation by proteolytic enzymes (proteases) and therefore Fab Production therefrom is difficult, chemical modification thereof, such as coupling thereof with an anticancer agent or a toxin, for instance, often results in loss of binding activity (J. W. Goding: Monoclonal Antibodies - Principles and practice, Academic Press, 1986).
  • monoclonal antibodies of the IgM class were superior in antitumor effect to monoclonal antibodies of the IgG class comparable to the former in reactivity for Thy-1 antigen-positive lymphocytes in an in vitro complement-dependent antitumor effect test while, in an in vivo antitumor effect test in cancer-bearing mice, monoclonal antibodies of the IgG class exhibited a significant antitumor effect but monoclonal antibodies of the IgM class did not show such effect.
  • glycolipids are important cell membrane constituents and readily undergo quantitative and qualitative changes upon canceration of cells [Cancer Res., 45 , 2405 (1985)]. Monoclonal antibodies to such glycolipids, if available, would be useful in the treatment and diagnosis of cancer and the effect of such monoclonal antibodies has actually been investigated in cancer patients [H, F. Oettgen (ed.): Gangliosides and Cancer, VCH Publishers, 1989].
  • attempts to produce monoclonal antibodies against non-T cell-dependent antigens, such as glycolipids readily lead to formation of IgM class monoclonal antibodies in most instances while IgG class monoclonal antibodies are hardly formed [A. H. Bartal and Y.
  • GM2 a ganglioside, which is a glycolipid containing a sialic acid residue, occurs only in very small amounts in normal cells but in large amounts in cancer cells, for example in the case of small cell lung carcinoma, melanoma or neuroblastoma, for instance, and therefore monoclonal antibodies to GM2 are thought to be effective in the treatment of such kinds of cancer [Lancet, 48 , 6154 (1988)].
  • monoclonal antibodies to GM2 that have been so far reported are of the IgM class [Cancer Res., 46 , 4116 (1986); Proc. Natl. Acad. Sci. U.S.A., 79 , 7629 (1982); Cancer Res., 48 , 6154 (1988); J. Biol. Chem., 264 , 12122 (1989)].
  • Whitmore et at. investigated as to what treatment could lead to high-frequency class switching of lymphoma B cells producing a monoclonal antibody of the IgM class against phosphorylcholine [Int. Immunol., 3 , 95 (1991)].
  • Whitmore et al. attempted to cultivate lymphoma B cells in the presence of interleukin-4, interleukin-5, interleukin-6, interferon- ⁇ , TGF- ⁇ , hydroxyurea, cholera toxin, or sheep erythrocytes (an unpurified antigen).
  • Hybridomas are said to change themselves spontaneously into hybridomas producing a class-switched monoclonal antibody, with a probability of one per tens of thousands to tens of billions [Eur. J. Immunol., 17 , 413 (1987)].
  • For preparing class-switched monoclonal antibodies it is thus necessary to select a hybridoma that has spontaneously changed in the above manner from among a great mass of unchanged hybridomas and proliferate it alone.
  • sib selection method which comprises cloning hybridomas using an agarose or 96-well plates and searching, one by one, for hybridomas that have changed [J.
  • the present invention provides a method of producing hybridomas capable of producing a monoclonal antibody of the IgG class which comprises cultivating a hybridoma producing a monoclonal antibody of the class IgM in the presence of an antigen reactive with said antibody and in the presence of thymocytes.
  • the invention also provides the hybridomas produced by the methods and antibodies produced by the hybridomas.
  • Fig. 1 shows the results of fluorescence intensity measurement by flow cytometry following reaction of FITC-anti-protein A with the hybridoma KM-697.
  • the ordinate is for the number of cells and the abscissa for the fluorescence intensity.
  • the solid line shows the pattern for the hybridoma reacted with FITC-anti-Protein A while the dotted line shows the pattern for the hybridoma not subjected to reaction with FITC-anti-protein A.
  • A shows the pattern for the hybridoma KM-697 free from antigen stimulation
  • B shows the pattern for the hybridoma KM-697 after five repetitions of antigen stimulation
  • C shows the pattern for the hybridoma KM-697 after six repetitions of antigen stimulation.
  • Fig. 2 shows the pattern of SDS polyacrylamide gel electrophoresis of a purified sample of the monoclonal antibody KM-750 (using a 4-15 gradient gel). From the left are shown the electrophoretic patterns of a molecular weight marker, a standard sample of IgG, a standard sample of IgM, and the monoclonal antibody KM-750, respectively.
  • A shows the results of electrophoresis performed under reducing conditions while B shows the results of electrophoresis performed under non-reducing conditions.
  • Fig. 3 shows the reactivities of some serially diluted (0.05-25 ⁇ g/ml) purified monoclonal antibodies for 20 pmol/well of GM2.
  • the symbol ⁇ is used for KM-750, o for KM-796, and ⁇ for KM-737.
  • Fig. 4 shows the reactivities of some purified monoclonal antibodies at a concentration of 10 ⁇ g/ml for serial dilutions (0.04-20 pmol/well) of GM2.
  • the symbol ⁇ is used for KM-750, o for KM-796, and ⁇ for KM-737.
  • Fig. 5 shows complement dependent cytotoxicity of the monocloanl antibodies against neurocytoma IMR32 which expresses ganglioside GM2.
  • the abscissa stands for the concentration of the antibody added.
  • the symbol ⁇ is used for KM-750 and o for KM-737.
  • the subject invention relates to hybridomas producing monoclonal antibodies of the IgG class which are produced by cultivating a hybridoma producing a monoclonal antibody belonging to the IgM class in the presence of an antigen reactive with the IgM antibody, and in the presence of thymocytes.
  • hybridoma producing a monoclonal antibody belonging to the IgM class there may be mentioned, for example, hybridomas producing a monoclonal antibody against a glycolipid such as the ganglioside GM2, sialylated Le x , Le x or Le y .
  • the hybridoma producing a monoclonal antibody against a glycolipid which is used as the antigen there may be mentioned, among others, the rat hybridomas KM-602, KM-603, KM-604, KM-605 and KM-606 and the mouse hybridomas KM-531, KM-693, KM-694, KM-695, KM-696 and KM-697, each capable of producing a monoclonal antibody reactive with the ganglioside GM2.
  • the hybridoma belonging to the IgM class used in the present invention can be prepared by production methods as conventionally used in preparation of known hybridoma strains.
  • the rat hybridomas KM-602, KM-603, KM-604, KM-605 and KM-606 and mouse hybridomas KM-531, KM-693, KM-694, KM-695, KM-696 and KM-697 can be prepared by the same method as described in JP-A-4-1138 or a modified one.
  • the hybridomas KM-531 and KM-603 have been deposited since September 14, 1989 and October 31, 1989, respectively, and KM-696 and KM-697 since April 2, 1991, with the Fermentation Research Institute, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan under the deposit numbers FERM BP-2597, FERM BP-2636, FERM BP-3337 and FERM BP-3338, respectively in accordance with the Budapest Treaty.
  • the thymocytes there may be mentioned mouse-derived thymocytes which mainly comprises T cells, macrophages and dendritic cells, and these are used at 1 x 106 to 1 x 108 cells/ml.
  • antigen stimulation is effected by adding an antigen reactive with the antibody produced by said hybridoma to the culture fluid.
  • the antigen concentration is 10 to 1 x 103 ng/ml, and the cultivation period required for one antigen stimulation is 5 to 7 days.
  • the antigen is added to the culture fluid preferably in a form encapsulated in liposomes.
  • the liposomes are prepared by using a phospholipid, such as dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylic acid, and cholesterol.
  • lipid A lipid A
  • lipid A lipid A
  • hybridomas producing a monoclonal antibody of the IgG class are isolated.
  • the sib selection method comprising cloning hybridomas using an agarose medium or 96-well plates and checking one by one in search of hybridomas that have changed [J. Immunol., 131 , 877 (1983)] and the method comprising using flow cytometry for collecting several hundred to several thousand hybridoma cells that have changed [J. Immunol. Methods, 52 , 1 (1982)] may be used.
  • the method involving flow cytometry is preferred, hence said method is described below in further detail.
  • FITC-anti-IgM fluorescent dye labeled with a fluorescent dye such as FITC (fluorescein isothiocyanate)
  • FITC-anti-IgM is reacted with hybridomas and the cell surface fluorescence intensity is measured by observation under a fluorescence microscope or by flow cytometric analysis, and hybridomas carrying a sufficient number of immunoglobulin molecules of the IgM class on the cell surface are selected.
  • a fluorescent dye-labeled anti-IgG antibody hereinafter, FITC-anti-IgG
  • fluorescent dye-labeled protein A hereinafter, FITC-protein A
  • fluorescent dye-labeled protein G hereinafter, FITC-protein G
  • the hybridoma cells obtained as described under (2) and showing a high fluorescence intensity are diluted to an appropriate cell density, distributed into wells of 96-well plates and cultivated.
  • the cell density should preferably be one cell per one well or per two wells (cloning by the limiting dilution method).
  • the cultivation should preferably be carried out in the presence of 1 x 104 to 1 x 107 thymocytes per milliliter.
  • the culture supernatants are sampled and the proportion of wells in which a monoclonal antibody of the IgG class is produced and the proportion of wells in which a monoclonal antibody of the IgM class is produced are examined by enzyme immunoassay, for instance.
  • the hybridomas in the wells in which a monoclonal antibody of the IgG class is produced are again cloned by the limiting dilution method.
  • the cloning by the limiting dilution method is repeated in that manner until wells in which a monoclonal antibody of the IgM class are no longer found and the production of a monoclonal antibody of the IgG class is detected in not less than 90% of the wells.
  • the hybridomas in those wells in which a monoclonal antibody of the IgG class is produced most abundantly are selected as class-switched hybridomas.
  • hybridomas producing a monoclonal antibody against the ganglioside GM2 such as KM-750 and KM-796.
  • the hybridomas KM-750 and KM-796 have been deposited, since April 2, 1991, with the Fermentation Research Institute, Agency of Industrial Science and Technology under the deposit numbers FERM BP-3339 and FERM BP-3340, respectively in accordance with the Budapest Treaty.
  • the class-switched hybridomas selected as described under (3) are cultured for 2 to 7 days using roller bottles or spinners. Each culture supernatant is collected by centrifugation and applied to a protein A column or a protein G column. The IgG class monoclonal antibody adsorbed is then eluted.
  • the monoclonal antibodies KM-750 and KM-796 produced against the ganglioside GM2 by the hybridomas KM-750 and KM-796, respectively. That the monoclonal antibody obtained belongs to the IgG class is checked by SDS electrophoresis, for instance.
  • pristane 2,6,10,14-tetramethylpentadecane
  • mice feeding them for 3 days to 2 weeks
  • intraperitoneally administering the hybridoma to them, collecting the resulting ascitic fluid therefrom, and purifying the antibody contained in the ascitic fluid using a protein A or protein G column.
  • the subclass of the monoclonal antibody can be determined by enzyme immunoassay using a subclass typing kit or by the Ouchterlony method which is based on immune precipitation.
  • the quantity of protein is determined by the Lowry method or by calculation based on the optical density at 280 nm.
  • the monoclonal antibodies according to the present invention can be used alone as an antitumor agent. They may be formulated into an antitumor composition together with at least one pharmaceutically acceptable carrier. For instance, the monoclonal antibodies are dissolved in physiological saline, an aqueous solution of glucose, lactose or mannitol and the like.
  • the powder of the monoclonal antibodies for injection can be prepared by lyophilizing the monoclonal antibodies in accordance with the conventional method and mixing the lyophilized products with sodium chloride.
  • the antitumor composition may further contain additives conventionally used well known in the art of medical preparation, for example, pharmaceutically acceptable salts.
  • the monoclonal antibodies according to the present invention can be administered in the form of the above-described antitumor composition to mammals including human in a dose of 0.2 to 20 mg/kg/day.
  • the dose may vary depending on the age, condition, etc. of patients.
  • the administration of the antitumor composition can be effected by intravenous injection once a day (single administration or consecutive administration) or intermittently one to three times a week or once every two to three weeks.
  • the antitumor composition is expected to be useful for treating tumors such as lung carcinoma, cerebroma and melanoma.
  • the rat hybridomas KM-602, KM-603, KM-604, KM-605 and KM-606 and the mouse hybridomas KM-531, KM-693, KM-694, KM-695, KM-696 and KM-697, each producing a monoclonal antibody of the IgM class which is reactive with the ganglioside GM2 were prepared according to the method described in JP-A-4-1138 and were examined for the presence or absence of an immunoglobulin on the cell surface.
  • KM-696 and KM-697 which showed high reactivity with the antigen and high antigen specificity, were selected for the subsequent experiments.
  • the monoclonal antibody KM-696 produced by the hybridoma KM-696 specifically reacted with N-acetyl-GM2 but did not react with other gangliosides such as N-acetyl-GM3, N-glycolyl-GM3, N-glycolyl-GM2, GM1, GD1, GD2, GD3, GD 1a , GD 1b , GT 1b and GQ 1b .
  • the monoclonal antibody KM-697 produced by the hybridoma KM-697 specifically reacted with N-acetyl-GM2 and N-glycolyl-GM2 but did not react with other gangliosides such as N-acetyl-GM3, N-glycolyl-GM3, GM1, GD1, GD2, GD3, GD 1a , GD 1b , GT 1b and GQ 1b .
  • GM2-containing liposomes were prepared in the following manner. In chloroform were dissolved 0.5 ⁇ mol of dipalmitoylphosphatidylcholine (Sigma), 0.05 ⁇ mol dipalmitoylphosphatidylic acid (Sigma), 0.5 ⁇ mol of cholesterol (Nakarai Tesque), 2.5 ⁇ g of lipid A (Funakoshi) and 2.0 ⁇ g of the ganglioside GM2 (Boehringer Mannheim). The solvent was then distilled off under reduced pressure to give a thin film. Thereto was added 0.5 ml of PBS. The vessel contents were heated at 50 to 60°C and then subjected to stirring with a mixer and 10 minutes of ultrasonic treatment to give GM2-containing liposomes.
  • a 24-well plate (Nunc), the several hundred thousand cells of each hybridoma as collected in the above manner were cultured in 1 to 1.5 ml of RPMI 1640 medium (Nissui Pharmaceutical) supplemented with 10% fetal calf serum (CSL) in the presence of mouse thymocytes (the number of cells being about one fiftieth of the total number of thymocytes of a 3- to 4-week old mouse).
  • antigen stimulation was effected by adding the GM2-containing liposomes to a final GM2 concentration of 500 ng/ml.
  • the cultivation for antigen stimulation in the presence of GM2-containing liposomes was conducted at 37°C for 6 to 7 days in the presence of 5% CO2. After antigen stimulation and when the number of hybridoma cells reached twenty to thirty million, the cells are reacted with FITC-anti-protein A, then washed with PBS, suspended in 6 ml of PBS and subjected to flow cytometry to thereby collect a group of several hundred thousand cells showing shifting to the higher fluorescence intensity side. After three repetitions of the cultivation for antigen stimulation followed by sorting by flow cytometry, the cultivation without antigen stimulation followed by sorting by flow cytometry was reheated two times for the hybridoma KM-696 and three times for the hybridoma KM-697.
  • Fig. 1 shows the fluorescence intensity pattern in flow cytometry of the hybridoma KM-697.
  • the samples were tested by the enzyme immunoassay technique mentioned below and the proportion of wells in which a monoclonal antibody of the IgG class was produced and that of wells in which a monoclonal antibody of the IgM class was produced were examined.
  • wells in which a monoclonal antibody of the IgM class were found. Therefore, the hybridomas in wells in which a monoclonal antibody of the IgG class was produced were again subjected to cloning by the limiting dilution method.
  • the hybridoma KM-750 was selected as a product of class switching from the hybridoma KM-697, and the hybridoma KM-796 as a product of class switching from the hybridoma KM-696.
  • GM2 (2 ng; Boehringer Mannheim), or 2 ng of any of other gangliosides, was dissolved in 2 ⁇ l of an ethanol solution containing 5 ng of phosphatidylcholine (Sigma) and 2.5 ng of cholesterol (Sigma). This solution or a dilution thereof was distributed in 20- ⁇ l portions into wells of a 96-well microtiter plate (Greiner) and, after air drying, blocking was effected using PBS containing 1% BSA. A hybridoma culture supernatant or a purified monoclonal antibody was added to the plate (50 to 100 ⁇ l per well) and the reaction was carried out at 4°C for 10 hours.
  • an ABTS substrate solution prepared by dissolving 550 mg of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in 0.1 M citrate buffer (pH 4.2) and adding, just prior to use, hydrogen peroxide in an amount of 1 ⁇ l/ml] was added to each well for color development.
  • the optical density at 415 nm (OD415) was then measured.
  • the hybridomas KM-750 and KM-796 were respectively cultured in 1 to 2-liter spinner incubators for 3 days to give culture supernatants in large amounts.
  • Each culture supernatant collected was applied to a protein A-Sepharose 4B column (Pharmacia) and the IgG class monoclonal antibody adsorbed was eluted with 0.1 M citrate buffer (pH 4.0). The eluate was dialyzed against PBS to give a purified monoclonal antibody.
  • SDS polyacrylamide gel electrophoresis proved that the purified monoclonal antibody was of the IgG class.
  • the gel used was a 4-15% gradient gel (Daiichi Kaguku). Fig.
  • FIG. 2 shows the results of SDS polyacrylamide gel electrophoresis of KM-750.
  • Antibody subclass determination by enzyme immunoassay using a mouse subclass typing kit (Zymed) revealed that the monoclonal antibody KM-750 produced by the hybridoma KM-750 and the monoclonal antibody KM-796 produced by the hybridoma KM-796 were both of the IgG3 subclass. Protein quantitation was carried out using a BCA reagent (Pierce).
  • the purified monoclonal antibodies were each tested, in serial dilutions (0.05 to 25 ⁇ g/ml), for reactivity with 20 pmol/well of GM2 by the ELISA technique.
  • the reactivity with GM2 of the monoclonal antibody KM-737 (of the IgG3 class) which is a monoclonal antibody against salmon growth hormone was also evaluated.
  • the monoclonal antibodies KM-750 and KM-796 both showed a high level of binding to GM2 in an antibody concentration-dependent manner, whereas the monoclonal antibody KM-737 was not bound to GM2.
  • the monoclonal antibodies KM-750 and KM-796 were tested for reactivity with the gangliosides GM1, N-acetyl-GM2 (Boehringer Mannheim), N-glycolyl-GM2, N-acetyl-GM3, N-glycolyl-GM3, GD 1a , GD 1b (Iatron), GD2, GD3, (Iatron), GT 1b (Funakoshi) and GQ 1b (Iatron) by the ELISA method.
  • GM1 and GD 1a were purified from the bovine brain, N-glycolyl-GM2 and N-glycolyl-GM3, from the mouse liver, and N-acetyl-GM3 was purified from canine erythrocytes and GD2 from the cultured cell line IMR32 (ATCC CCL127), essentially by a per se known method [J. Biol. Chem., 263 , 10915 (1988)].
  • the results are shown in Table 1.
  • Table 1 As shown in Table 1, KM-750 reacted only with N-acetyl-GM2 and N-glycolyl-GM2 but did not react with the other gangliosides.
  • This reaction specificity is the same as that of the monoclonal antibody KM-697 before class switching, indicating that the reaction specificity remained unchanged even after class switching to IgG.
  • KM-796 reacted only with N-acetyl-GM2, but did not react with the other gangliosides.
  • This reaction specificity is the same as that of the monoclonal antibody KM-696 before class switching and thus, for KM-796 as well, the reaction specificity remained unchanged even after class switching to IgG.
  • Neurocytoma IMR32 (ATCC CCL 127) which expresses the ganglyoside GM2 was suspended in the RPMI1640 medium supplemented with 10% fetal calf serum (FCS) and adjusted to a cell density of 1 ⁇ 107 cells/ml to serve as target cells.
  • FCS fetal calf serum
  • Na251CrO4 was added thereto to 100 ⁇ Ci/l ⁇ 107 cells and the medium was incubated at 37°C for 1 hour.
  • the cells were washed with the RPMI1640 medium supplemented with 10% FCS three times.
  • the whole cells were allowed to stand at 4°C for 30 minutes in the RPMI1640 medium supplemented with 10% FCS for dissociation. After centrifugation at 1,200 rpm for 10 minutes, the cell density was adjusted to 4 ⁇ 106 cells/ml by adding the RPMI1640 medium supplemented with 10% FCS.
  • the target cells prepared in above (a) (2 X 107 cells) were added to 1 ml of rabbit complement (Cederane Laboratory) and allowed to react at 4°C for 1 hour. The cells were filtered off to give a complement solution.
  • the monoclonal antibodies KM-750 and KM-737 were each added to the wells of the 96-well U-bottom plate to a final concentration ranging within 0.05 to 50 ⁇ g/ml and further 2 ⁇ 105 cells of the target cells were added to each well. After a reaction at room temperature for 1 hour, centrifugation was carried out at 1,200 rpm for 5 minutes to remove the supernatant.
  • the complement solution prepared in above (b) was 9-fold diluted with the RPMI1640 medium supplemented with 10% FCS and a 150 ⁇ l portion of the dilution was added to each well. A reaction was carried out at room temperature for 1 hour and then the coils were removed by centrifugation at 1,200 rpm for 5 minutes.
  • the amount of 51Cr in 75 ⁇ l of the supernatant was determined by a ⁇ -counter.
  • the amount of naturally dissociated 51Cr was determined in the same manner as described above except that the RPMI1640 medium supplemented with 10% FCS alone, containing neither monoclonal antibody nor complement, was added to the target cells.
  • the total amount of dissociated 51Cr was determined in the same manner as described above except that 5N sodium hydroxide solution in place of the monoclonal antibody and the complement solution was added to the target cells.
  • CDC activity was calculated by the following equation.
  • the present invention provides a method of producing hybridomas which comprises subjecting hybridomas producing a monoclonal antibody of the IgM class to class switching to hybridomas producing a monoclonal antibody of the IgG class.
  • the monoclonal antibodies of the IgG class produced by said hybridomas can be used widely in the treatment of various diseases.

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EP92106282A 1991-04-10 1992-04-10 Verfahren zur Herstellung von Hybridomen Expired - Lifetime EP0508472B1 (de)

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JP07751891A JP3150991B2 (ja) 1991-04-10 1991-04-10 ハイブリドーマの製造法
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU669379B2 (en) * 1992-09-07 1996-06-06 Kyowa Hakko Kirin Co., Ltd. Humanized antibodies
EP2242836A4 (de) * 2008-01-28 2011-05-25 Univ Jefferson Verfahren zur herstellung von hybridzellen zur expression nützlicher antikörper

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0789590B1 (de) * 1994-09-23 2002-09-04 Zonagen, Inc. Chitosan induzierte verstärkung
US5912000A (en) * 1994-09-23 1999-06-15 Zonagen, Inc. Chitosan induced immunopotentiation
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AU669379B2 (en) * 1992-09-07 1996-06-06 Kyowa Hakko Kirin Co., Ltd. Humanized antibodies
US5874255A (en) * 1992-09-07 1999-02-23 Kyowa Hakko Kogyo Co., Ltd. DNA encoding humanized antibodies specific for the ganglioside GM2
EP2242836A4 (de) * 2008-01-28 2011-05-25 Univ Jefferson Verfahren zur herstellung von hybridzellen zur expression nützlicher antikörper
US8999707B2 (en) 2008-01-28 2015-04-07 Thomas Jefferson University Method of making hybrid cells that express useful antibodies

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JPH04311385A (ja) 1992-11-04
EP0508472A3 (en) 1993-08-04
CA2065681A1 (en) 1992-10-11
JP3150991B2 (ja) 2001-03-26
DE69233122T2 (de) 2004-04-22
DE69233122D1 (de) 2003-08-14

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